Neurotransmitter release is acutely triggered by Ca2+ and is regulated during presynaptic plasticity processes that underlie some forms of information processing in the brain. Characterization of the mechanisms of release and its regulation is thus critical to understand brain function and will facilitate the development of therapies for neurological disorders with a presynaptic origin. The machinery that controls release contains a core formed by Munc18-1 and SNARE proteins, and specialized proteins that regulate release. Several of these proteins contain multiple C2 domains, which are widespread Ca and phospholipids binding modules but can 2+ also exhibit Ca2+-independent activities. These proteins include: i) Synaptotagmin-1, the Ca2+ sensor that triggers fast release; ii) Munc13-1 and related isoforms, which are essential for release and mediates diverse forms of presynaptic plasticity; and iii) RIMs, which are Rab3 effectors that also have key roles in release and presynaptic plasticity. The C2 domains of all these proteins are highly conserved and are hypothesized in this proposal to regulate neurotransmitter release at multiple levels through their Ca2+-dependent and Ca2+- independent interactions. The ultimate goals of the research proposed in this application are to test this hypothesis and, more generally, to elucidate the molecular mechanisms underlying key forms of regulation of neurotransmitter release. This research forms part of an integrated approach where the structural and reconstitution data performed in the PI's lab are correlated with genetic and physiological experiments performed in the laboratories of close collaborators. We propose three Specific Aims that focus on the following areas: 1. Mechanism of action of Synaptotagmin-1; 2. Structural basis for Munc13-dependent regulation of neurotransmitter release; and 3. Reconstitution of Munc13-dependent regulation of neurotransmitter release. The proposed experiments will build on exciting results obtained during the previous funding period, including a preliminary structure of a Synaptotagmin-1/SNARE complex, key advances in crystallization of large Munc13-1 fragments, the reconstitution of synaptic vesicle fusion with the eight most central components of the release machinery, and preliminary data that reinforce the notion that Munc13s act as master regulators of release through a fascinating network of intramolecular and intermolecular interactions. We expect that this research will provide critical insights into the regulation of release in varied presynaptic plasticity processes, helping to establish fundamental principles on neuronal communication that are vital for brain function.